Adding His tags to PCR products

Maria Cristina Costanzo mcc9 at cornell.edu
Tue Sep 12 11:10:27 EST 1995


In article
<Pine.A32.3.91j.950908105409.81303A-100000 at homer16.u.washington.edu>,
Mayumi Yagi <myagi at u.washington.edu> wrote:

> Hi, netters,
> 
> I've been having a major problem with something I thought would be 
> relatively easy: Adding an enterokinase site and His tag to a product 
> cloned by PCR.  I have a 1kb cDNA fragment cloned into PCR3 that I 
> thought it would be nice to have in a form I could purify.  So, I decided 
> to use this clone as a template for PCR using an oligo that has the last 
> 9 or so codons, an enterokinase site (Asp4-Lys-Glu), a couple more AA, 
> then His6, termination codons, and an RE site.  I got TONS of PCR 
> product, which I then recloned into PCR3 and sequenced.  Result: all of 
> the clones had various deletions/rearrangements in the EK/His oligo.

I added 6XHis tags to 3 different yeast genes using this method with no
problems whatsoever. My oligos didn't have an enterokinase site, however -
maybe something about this sequence is causing the problem?
 
Maria Costanzo



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